Elisa screening method for inhibitors of human IgE binding to the high affinity receptor, FcεRIα

ABSTRACT

The present invention is directed to an Assay for high capacity screening of substances interfering with the attachment of human IgE to its high affinity receptor and/or of substances capable of detaching already bound IgE from this receptor and for the differential analysis between autoimmune disorders and classical allergies.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a 371 of PCT/EP96/05824, filed Dec. 23, 1996.

FIELD OF THE INVENTION

The invention relates to a test system by ELISA (enzyme-linkedimmunosorbent assay). It concerns an ELISA system to detect biochemicalentities in very minute amounts and in the presence of structurally verysimilar compounds, which are differentiated by means of monospecificantibodies, such as immunoglobulins of various species.

DESCRIPTION OF THE RELATED ART

In conventional ELISA systems either the antigen which is to bedetected, or a specific antibody which binds to the respective antigen,is bound to a solid phase (microtiter plate) by hydrophobicinteractions: the protein interacts with the solid phase, usually apolystyrene surface at high pH. Although this bond is responsible forall the consecutive steps in the procedure, it remains the weakestbridge to the assay support, the ELISA plate. Strong detergents athigher concentrations such as 0.05% are able to diminish the amount ofreagent bound to the plate and can even abolish binding totally. Theconsecutive steps in ELISA technique, such as the binding of an antigento the solid phase-bound antibody and further binding of a secondantibody, occur with an affinity of approximately 10⁻¹² to 10⁻¹⁰ mol perliter. One can view this as being similar to an inverse binding cascadefrom the bottom of the plate to the top, as with a pyramide standing onits top.

Another concern is the intramolecular event upon binding. A protein suchas a cell-receptor or an antibody behaves very flexibly according to itspolypeptide structure, which forms, a complex architecture in solution.This explains its high specificity and selectivity to the ligands whichare to bind to it in vivo. Enzymatic activities for example are entirelydependent upon the proper formation of the active site pocket, whichitself remains flexible in order to engulf the substrate and release theproduct. Cell-receptors such as FcεRIα react with an affinity for IgE invivo of 10⁻¹⁰ mol per liter provided the respective active site isproperly exposed. Most proteins react to binding to a given surface witha dramatic change of their tertiary structure, i.e. they unfold, refold,hide their active site or change their conformation in such a way thattheir reactivity toward a given ligand is altered or even cancelled. Inorder to circumvent this disadvantage, in conventional ELISA systems acatching antibody is used. This antibody binds to the polystyrene plateand exposes the high affinity hyper-variable region toward the incomingantigen. The antigen is then detected by a second antibody, which islabelled directly or indirectly (via biotin/avidin) with an enzyme. Thisenzyme will cleave a chromogenic substrate, which itself is convertedfrom the leucoform to the chromoform and thus visualizes the presence ofthe antigen in question. But even catching antibodies may affect a givenprotein by changing its conformation. This is demonstrated by manyexamples of therapeutic antibodies whose mode of action is the blockingof an active site on, or the alteration of, a biospecific molecule.

SUMMARY OF THE INVENTION

The present invention concerns an assay for high capacity screening ofsubstances interfering with the attachment of human IgE to its highaffinity receptor FcεRIα, and/or of substances capable of detachingalready bound IgE from this receptor, which is devoid of the abovedisadvantages. It comprises reacting a solution of a biotinylated FcεRIαreceptor with IgE, transferring the resultant binding reaction mixtureto a streptavidin-coated plate and quantifying by means of anappropriate enzyme-labeled antibody. More specifically, it comprisesreacting biotinylated IgE-receptor with IgE in the presence or absenceof interfering or inhibitory substances, such as non-biotinylatedIgE-receptor at well-defined molar concentrations, temperature, pH andsalt conditions, thereby eliminating the structural influence of solidphases, and transferring after a defined incubation time an aliquot ofthe binding reaction mixture to a streptavidin-coated multiple wellplate, where the biotinylated IgE-receptor/IgE complex is quantified bymeans of an appropriate enzyme-labeled antibody, such as horse radishperoxidase (POD)-labeled antibody against human IgE.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The principle of this assay method is as follows: biotinylatedIgE-receptor is first reacted with IgE in the presence or absence ofinterfering or inhibitory substances, such as non-biotinylatedIgE-receptor, at well-defined molar concentrations, temperature, pH andsalt conditions, thereby eliminating the structural influence of solidphases. After a defined incubation time an aliquot of the bindingreaction mixture is transferred to a streptavidin-coated multiple wellplate, where the biotinylated IgE-receptor/IgE complex is trapped andquantified by means of an appropriate enzyme-labeled antibody, such ashorse radish peroxidase (POD)-labeled antibody against human IgE.

During the reaction of the receptor with its ligand (IgE) in solution,both molecules float freely in solution and thus are not influenced bythe structural modification of solid phases. They receive theirconformation by the given pH and salt concentration and exert theirmaximal binding affinity. After a given incubation time, selectedaccording to the results of appropriate equilibrium measurements, thereceptor/ligand complex has to be deprived of all other compounds. Thereceptor is therefore biotinylated prior to the above reaction and analiquot of the reaction is transferred to a streptavidin-coated multiplewell plate. In order to build up an affinity cascade from the bottom ofthe multiple well plate to the top of the reactants, the binding ofstreptavidin with the plate surface is preferably made covalent. Forexample, a microtiter plate (such as a DNA-Bind plate) chemicallymodified with e.g. a N-oxysuccinimide ester coating is used, whichreacts with nucleophiles such as primary amines under formation of acovalent bond. This bond is stronger than 10⁻¹⁵ moles per liter. For thenext step, the biotinylation of the receptor, a chemically modifiedbiotin, such as NHS-LC-Biotin is used, which comprises an extendedspacer arm of approximately 22.4 Å in length. This long chain analoguereduces steric hindrances associated with the binding of fourbiotinylated molecules on one streptavidin molecule. The target ofbiotinylation on the IgE-receptor molecule is thereby well defined andinterference with the ligand is avoided. The binding affinity of biotinto streptavidin is also known to be 10⁻¹⁵ moles per liter. Thereby thefirst two steps of the binding cascade are established and reach fromthe bottom of the plate to streptavidin to the biotinylated receptor.The affinity constants are decreasing from the bottom to the top. Atthis stage the reactants, which have found their partners in solutionunder appropriate conditions, are trapped by the means of thestreptavidin plate, and the bound IgE is then detected with anappropriate enzyme-labeled antibody, such as POD-labeled antibodyagainst human IgE. The affinity cascade now reaches from <10⁻¹⁵ to 10⁻¹⁵to 10⁻¹⁰ to 10⁻¹² for the anti-IgE antibody toward IgE. Any interferenceof biochemical material and especially of IgE-receptor bindinginhibitors will occur at the point with the lowest affinity, whichresides between the IgE and the receptor (10⁻¹⁰), and not at the pointof attachment of the assay to the plate. This would be read as a falsepositive result—an artifact of the system.

The above provides the basis for the detection of biomolecules otherthan IgE but which are also binding to FcεRIα. The prerequisite thereforis the abolishment of the binding of IgE to the receptor in order tokeep the receptor site free for other molecules. This is achieved bymeans of an anti-IgE antibody as BSW17 which binds to thereceptor-binding site of the IgE and thus renders the moleculenon-effective, whereby even in the presence of IgE other biomoleculescan bind to the IgE-binding site of the receptor (the hybridoma cellline producing monoclonal antibody BSW17 has been deposited on Dec. 18,1996 with the European Collection of Animal Cell Cultures (ECACC),Porton Down, U.K. under the provisions of the Budapest Treaty on thedeposit of microorganisms, under deposit No. 96121916).

More particularly, the invention concerns an ELISA-type assay forfinding inhibitors of the binding of human IgE to its high affinityreceptor, the α-chain of FcεRI (FcεRIα), especially substancesinhibiting IgE-mediated early phase response in allergic reactions. Theassay mimics natural conditions of binding of IgE to its receptor, i.e.all binding partners interact in solution instead of one of them beingimmobilized on a solid support. In the binding reaction biotinylatedsoluble FcεRIα chain interacts with free, soluble IgE at a defined molarratio (1:2.5) in the presence of inhibitory or interfering substance toreach thermodynamic equilibrium within a three-hours incubation periodat 37° C. The IgE/FcεRI complex formed under these conditions iscaptured via the biotin-linker coupled to the FcεRIα chain on ELISAplates modified by covalently-linked streptavidin. The amount ofcaptured complex is determined by identifying bound IgE via aperoxidase-linked anti-human IgE antibody preparation, such asanti-human IgE (ε-chain-specific) peroxidase conjugates e.g. Sigma no.A-9667. The concentrations of the binding partners in primary screeningare chosen to obtain an optimal signal to noise ratio undernonsaturating conditions. In this experimental setting the K_(d) of theIgE/FcεRI complex formed is found to be 4 nM. The maximally tolerateddose of DMSO in the test solution is 5% and therefore, at aconcentration of pure compound of 50 μM competitive inhibitors with aK_(d) of 30-10 μM can be easily detected in primary screening at acut-off level of 30% inhibition. As a standard control a monoclonalantibody recognizing free as well as receptor-bound IgE and inhibitingits binding to the receptor or releasing it from the complex,respectively, such as BSW17, is tested in 5 different concentrations onevery screening plate.

Some of the advantages of this assay in comparison to standard ELISAsystems where the receptor or antigen first will be attached to a solidsupport with either a catching antibody or by high pH (9.6) are thefollowing:

1. The structural influence by the supporting solid phase or thecatching antibody in standard sandwich-ELISA is avoided;

2. no treatment of the receptor or antigen at alkaline pH;

3. defined molar concentrations of the reaction partners; and

4. reduced working steps and easy handling by robotics.

Allergies in their major manifestations, known as asthma, allergicrhinitis or atopic dermatitis, afflict more than 20% of the populationin industrialized countries. They are generally caused by anoverproduction of IgE in genetically predisposed individuals in responseto common environmental antigens. The high affinity receptor for IgE,the multimeric FcεRI present on mast cells, basophils, human Langerhanscells and monocytes of allergic individuals, mediates immediatehypersensitivity responses if aggregated by IgE/allergen complexes.Therefore, to interfere with the binding of IgE to FcεRI is a major newstrategy for alternative treatment of allergic patients. Severalapproaches in this direction are already in early clinical evaluation orin preclinical development, such as the use of blocking antibodiesagainst IgE, of recombinant soluble FcεRI α-chain, or of inhibitorypeptides. In view of the well-known drawbacks of such approaches, suchas potential immunogenicity, poor bioavailability and high treatmentcosts, low molecular weight compounds inhibiting this crucialinteraction in allergic response are needed. As the affinity of IgEtoward FcεRI is high (K_(a)=10¹⁰ M⁻¹) a low hit-rate in any samplecollection is expected. However, the throughput of test samples in theabove assay can be very high (>1000 samples/day) and the sensitivity isalso high, as compounds which competitively inhibit the binding reactionwith a K_(d) of 10 μM are easily detected by a signal reduction of 50%.

Further, the soluble extracellular part of the α-chain, the IgE-bindingsubunit of FcεRI, is available through recombinant DNA technology andhence the assay for high capacity screening of inhibitors of theIgE/FcεRI interaction could be established cell-free. To mimic thenatural binding reaction of IgE to the α-chain the assay is designed forthe interaction of binding partners to occur in solution. Instead ofdirectly immobilizing one binding partner on a solid support bynon-covalent adsorption techniques, e.g. the α-chain by hydrophobicinteractions on the surface of ELISA plates, an interaction which ishighly susceptible to detergent-like substances, a more stable form offixation to the solid medium is evaluated. The affinity of biotin forstreptavidin is orders of magnitude higher (K_(d) approximately 10⁻¹⁵ M)than that of IgE for FcεRI. Therefore, inhibitors of the IgE/FcεRIinteraction are unlikely to disturb the interaction of biotin andstreptavidin. Additionally, the latter is insensitive to extremes of pH.Hence, this interaction was chosen to capture the IgE/α-chain complex onthe surface of ELISA plates. For this purpose, the α-chain isbiotinylated using an extended spacer arm for chemical coupling andstreptavidin is covalently bound to the ELISA plate via amino groups.The binding reactions between IgE and its high affinity receptor,FcεRIα, and the interference of test compound are performed in aseparate multiple well plate to reach the equilibrium in solution duringan extended incubation period at 37° C. To favor conditions forinhibitory compounds to be active, they are added to and preincubatedwith the α-chain prior to the addition of IgE. The residual IgE/α-chaincomplex formed in the presence of test compound is then captured on thestreptavidin plate during a short incubation period at room temperatureor alternatively, after an overnight incubation at 4° C. The amount ofcaptured complex is determined by a peroxidase-catalyzed colorimetricreaction using an anti-human IgE antibody preparation with covalentlycoupled enzyme.

The key steps of the assay are schematically presented in FIG. 1. Thefollowing Example illustrates the invention but is not limitativethereof.

EXAMPLE

Assay Procedure

1. Preparation of Covalently-Bound Streptavidin Microtiter Plates

To 12 ml of PBSdef. PH9, 6 μl of streptavidin solution is added and themixture is stirred for a few minutes [1 μg/ml]. 100 μl of this solutionare pipetted into each well of a Costar Amine plate, which is takendirectly from 4° C. storage and dismantled from its protective envelopeprior to pipetting. The whole procedure is performed in the dark and theplate must be kept in a light protective aluminium foil for 1 hour atroom temperature. Thereafter, the consecutive steps can be done undernormal conditions: five washes (Biomek) of the plate with PBST andtapping onto a paper towel in order to free the plate from residualmoisture, addition of 360 μl of 0.5 M Tris-Cl pH 8 and incubation for 1hour at room temperature. This step will inactivate residual free estergroups on the plate surface. Then, 200 μl of blocking solution (2% BSAin PBS, 0.05% Tween 20) is added into each well and kept at roomtemperature for 30 minutes. Then the plate is washed once more asdescribed and stored in a sealed plastic box at 4° C.

2. Biotinylation of the FcεRIα Receptor

A given amount of receptor solution (FcεRIα, extracellular portion,recombinant, produced in insect cells) is concentrated byultrafiltration over Amicon YM 3/25 to give 1.5 ml at a concentration of2.2 mg protein per ml. This solution is dialyzed with a PierceSLIDE-ALYZER against three times 1 liter of 100 mM sodium carbonate pH8.5 within four hours at 4° C. The biotinylation is done by adding 23 μlof a solution of 4.8 mg of Biotin II Pierce in 120 μl of DMF and thevial is tumbled for 15 minutes at room temperature. After anotheraddition of 23 μl of the biotin solution the whole device is transferredto 4° C. and tumbling continued for 1.5 hours. Then the reaction isstopped by addition of 150 μl of 1 M Tris-Cl pH 8 and a furtherincubation for 10 minutes at room temperature.

3. Column-Chromatography

This sample is loaded onto a FPLC Superdex 75 [16×1200 mm] in PBS 0.02%sodium azide at 4° C. The flow-rate is 2 ml/min, the fraction size is2.6 ml. The fractions containing adequately biotinylated receptor arepooled and concentrated over an Amicon YM 3/43 to approximately 4 ml.After determination of the protein concentration an equal volume ofglycerol is added. 100 μl aliquots of this solution are stored at −80°C. 50 μl of fraction aliquots are placed into each well of astreptavidin-covalent-plate and kept at room temperature for one hour.Thereafter the plate is washed with PBST and 50 μl of IgE [20 nM] areadded to each well and incubated for one hour at room temperature. Aftera subsequent wash with PBST, 100 μl of anti-IgE-POD [1:1000] are addedto each well and the plate is further incubated for one hour. Then theplate is washed again and 100 μl of TMB substrate are added. After 20minutes of incubation at room temperature the reaction is stopped byaddition of 100 μl of 4 N H₂SO₄ and the plate is read at 450 nm.

A high molecular weight pool is detected containing highly biotinylatedbut aggregated material, which binds to the plate but consequently isinactive in binding to IgE. The appropriate IgE-binding competentmaterial is found at the expected elution position of 32 kDa. Remainingreactants are seen in a low molecular weight fraction as well.

4. Screening

All substances to be tested are distributed into multiple well platesfor testing in solutions of 10 mM NaCl; 50% DMSO at a concentration of200 μg/ml or 500 μM. These solutions in conical bottom plates arediluted once more 1:3.3 (20 μl of substance plus 46 μl of incubationbuffer) into round-bottom plates to give 66 μl of 150 μM substance in15% DMSO. These plates are adjusted in position A4-A12 by emptying thewells by suction (the whole line A1-A3 is dedicated for mediumreferences=High control) and 2 M NaOH is pipetted into wells A9-A12 inorder to give the Low control. IgG BSW17 is pipetted into well A4-A8 at12 nM in two-fold dilutions. These are the dilution plates. Then theworking solution for the receptor is prepared by diluting the stocksolution to 600 pM in incubation buffer. 50 μl of this solution isdistributed to each well of a new round bottom plate (incubation plate)and 50 μl of the respective dilution plate is transferred to this plateand mixed with the receptor five times. The plate is kept for one hourat 37° C. covered with an adhesive foil and thereafter 50 μl of IgE B11at a concentration of 1500 pM is pipetted into each well of theincubation plate and mixed five times. The respective concentrations areas follows: 5% DMSO, 50 μM substance, 600 mM NaOH, 200 pM receptor, 500pM IgE; 4; 2; 1; 0.5; and 0.25 nM IgG BSW17.

The plate is sealed with an adhesive foil and incubated for three hoursat 37° C. Thereafter 100 μl are transferred from the incubation plateinto the streptavidin covalent plate which is kept overnight at 4° C.After a five times wash 100 μl of anti IgE-POD [1:2000] are added andthe plate is incubated for another hour at room temperature. The plateis washed again five times and 100 μl of TMB-substrate are added. After20 minutes incubation at room temperature the reaction is stopped byaddition of 100 μl of 4 N H₂SO₄ and the plate is read at {fraction(450/690+L )} nm.

Results:

FIG. 2 depicts the functional properties of the FcεRIα receptor at 20 nMIgE. The receptor concentration of 200 pM chosen for the screening assayrepresents the high endpoint of the exponential phase of the curve.Previous experiments showed that the molar binding ratio for IgE is1:2.5 and hence 500 pM was chosed as the assay concentration for IgE.The IgG BSW17 standard provides a measure for the sensitivity of theassay system as well as a gauge for the determination of the IC50.

Abbreviations:

B11=recombinant human IgE monoclonal antibody (protein concentration1850 μg/ml; molecular weight 188 kDa)

BSA=bovine serum albumin

BSW17=IgG monoclonal antibody directed against the CH₃ epitope of nativeIgE (protein concentration 2.3 mg/ml; molecular weight 150 kDa)

CU=chronic urticaria

DMF=dimethylformamide

DMSO=dimethylsulfoxide

EDTA=ethylene diamine tetraacetic acid

ELISA=enzyme-linked immunosorbent assay

FCS=fetal calf serum

FPLC=forced pressure liquid chromatography

IgE=immunoglobulin E

LC=long chain spacer arm

mAb=monoclonal antibody

Mwt=molecular weight

PBS=phosphate-buffered saline

PBSTi=phosphate-buffered saline/Tween 20

POD=horse radish peroxydase

TMB=tetramethylbenzidine

WB=Western blot

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Key steps of assay:

1. Couple streptavidin covalently onto ELISA plates

2. Mix biotinylated FcεRIα-chain and substance solution

3. Incubate

4. Add IgE, incubate and transfer preformed complex onto ELISA plate

5. Wash the plate free of unbound compounds

6. Add detecting antibody (peroxidase-linked anti-human IgE) andincubate

7. Wash and develop with peroxidase substrate

FIG. 2: Binding capacity of FcεRIα at 20 nM IgE

▪=IgE binding

FIG. 3: Scheme of the anti-FcεRIα ELISA.

I) Serum specimens are reacted with an anti-IgE mAb and thereafterincubated with biotinylated rsFcεRIα;

II) IgE is blocked, α-chain specific IgG can bind to its antigen;

III) IgG anti-FcεRIα/rsFcεRIα_(biot) complexes bind to thestreptavidin-coated plate and are detected with enzyme-coupledanti-human IgG.

FIG. 4: mAb BSW17 inhibits IgE binding to rs FcεRIα.

Sera (diluted 1:15) from atopic patients (AD) and from chronic urticariapatients (CU) were analyzed for anti-IgG and anti-IgE reactivity withrsFcεRIα in the presence (closed bars) or absence of the anti-IgE mAbBSW17 (dotted bars). y-axis shows dilution-corrected optical density(OD×1000).

=serum; ▪=serum reacted with anti-IgE (BSW17).

FIG. 5:

(A) Preincubation of streptavidin-coated plates with anti-FcεRIαnon-reactive sera does not block the reactivity of IgG anti-FcεRIαreactive sera. Reactivity of two positive and one negative specimen isshown.

(B) Increasing concentrations of randomly selected serum do not affectthe detectability of anti-FcεRIα autoantibodies.

(C) Reactivity of autoantibodies to biotinylated rsFcεRIα is blocked bypreincubation with unlabeled rsFcεRIα protein but not by preincubationwith rhesus monkey papilloma virus (rRhPVL.1).

FIG. 6: Unlabeled rsFcεRIα-chain protein was added up to 100-fold higherconcentration than the biotinylated receptor (x-axis). Results withserum from anti-FcεRIα-reactive and non-reactive chronic urticariapatients (CU), from atopic dermatitis patients (AD) and from healthycontrols (CO) are depicted. The y-axis shows the optical densitymultiplied by the dilution factor.

FIG. 7: Correlation of anti-FcεRIα reactivity in Western blot analysisand ELISA. Western blot-reactive (CU pos), Western blot non-reactive (CUneg), and sera that could not be defined by blotting analysis (CU nd)were analyzed in 1:100 (x-axis, OD×100) and 1:400 (y-axis, OD×400)dilution.

FIG. 8: Sera from 85 chronic urticaria patients (CU), 20 atopicdermatitis patients (AD) and 31 healthy controls (CO) were analyzed forthe presence of anti-FcεRIα autoantibodies by ELISA. Reactivity isdefined as optical density at a 1:400 dilution (x-axis) versus thersFcεRIα-blockable reactivity (y-axis).

Materials:

TMB stock solution: 2 mg TMB/ml ethanol, filtered through a glass sinterfilter no. 2;

TMB working solution; 100 ml citrate buffer pH 5 plus 2.2 ml TMB stocksolution plus 560 μl 0.3% H₂O₂:

Wash buffer: PBSdef. with 0.05% TWEEN 20;

Blocking solution: Wash buffer with 2% BSA;

Incubation buffer: Wash buffer with 2% FCS;

rec human IgE receptor: R1α[470 μg/ml] EN 24/460, Mwt 32 kDa;

human IgE: B11 [1850 μg/ml]; Mwt 188 kDa;

biotinylated human IgE receptor: FcεRIα-LC-Biotin Pool 5 [120 μg/ml] Mwt32 kDa;

PBSdef. pH 9: PBSdef. titrated to pH 9 with 2 M NaOH;

Citrate buffer pH 5: 10 mM (0.86 g citric acid+1.74 g sodium citrate perliter); 10 mM EDTA (3.7 g per liter);

Stop solution: 4 N sulfuric acid;

Streptavidin solution: 2 mg/ml distilled water;

IgG BSW17 [2.3 mg/ml] Mwt 150 kDa.

FURTHER DESCRIPTION OF THE PREFERRED EMBODIMENTS

In a preferred embodiment of the invention this assay method is usefulfor the differential analysis between autoimmune disorders and classicalallergies, i.e. allergies triggered by exogenous factors.

Urticaria is a common disorder characterized by the eruption oftransitory, itchy skin swellings that are frequently associated with theoccurrence of debilitating and potentially life-threatening angioedema.Although accurate data on the prevalence of urticaria are not available,it is estimated that 15% to 32% of the general population experienceurticarial/angio-edematous symptoms during their life time. In about 25%of these patients the initially sporadic character of this condition isfollowed by a chronic disease period with symptoms of frequent butunpredictable occurrence. If widespread wheals tend to appear daily oralmost daily for at least six weeks the disease is termed chronicurticaria (CU). Taken together, CU is a frequent disease afflicting aconsiderable proportion of the population in the Western hemisphere.

Although it is a well known fact that histamine release from mast cellsis of central pathophysiological relevance for the elicitation of CU,the causative agent(s) that mediate mast cell activation remainedobscure until very recently. The existence of autoantibodies directedagainst the high affinity IgE receptor FcεRI in the serum of CU patientswas shown. These autoantibodies were found to cross-link FcεRI onbasophils and mast cells, resulting in the exocytosis of histamine bythese cells. Using recombinant soluble FcεRIα as a reaction target forautoantibodies, it was found that about 37% of CU sera exhibited IgGautoreactivity against FcεRIα. No IgG anti-FcεRIα reactivity wasobserved in the serum of atopic dermatitis patients or healthy controlindividuals. Therefore, IgG autoreactivity against FcεRIα is of centralpathophysiological importance for CU and furthermore is a selectivemarker identifying an autoimmune-mediated sub-entity of this disease. Itis hence of great interest to develop strategies that allow the reliableand fast detection and quantification of such serum autoantibodies.

A further indication of the urgent need for an appropriate screeningsystem comes from the fact that up to now laborious and cost-intensivesearches for certain implicated, but not unequivocally proven,etiopathogenetic conditions, e.g. bacterial or fungal infections,hormonal dysregulation, psychological factors, neoplasms, andintolerance to certain food activities. are performed in most CUpatients. It is obvious that such laboratory, physical, andpsychological examinations, which often require long-lastinghospitalization of the patients. cause enormous financial expense. Thus,the quick and easy detection of disease-eliciting autoantibodies helpsto drastically reduce the requirement for diagnostic procedures that areunrevealing in most patients.

Design of an Anti-FcεRIα ELISA System:

1. Expression and Purification of Human Recombinant Soluble FcεRIα

The gene segment encoding the extracellular portion of human FcεRIα iscloned into the baculovirus vector pVL941 (BACULOGOLD Transfection Kitno. 21100 K, Baculovirus Vector PVL1392/1393 no. 21201 P, PharMingen,D-22335 Hamburg, Germany). Recombinant baculovirus is generated ininsect cells using the BACULOGOLD transfection kit (PharMingen)according to the manufacturer's instructions. The soluble receptormolecule is purified from supernatants of infected insect cells by a twostep chromatography protocol. First, the material isimmunoaffinity-purified on a Sepharose 4B (Pharmacia Biotech)—coupledmouse anti-human FcεRIα mAb (E. Fiebiger et al., J. Clin. Invest. 96[1995] 2606-2612). Final purification is achieved by anion exchangecolumn chromatography. The purification product migrates as a single 32kD band as judged by silver staining of a denaturing polyacrylamide gel.

2. Insect Cell-Expressed Recombinant Soluble FcεRIα (rsFcεRIα) as aReaction Target for IgG Anti-FcεRIα Autoantibodies

Solid-phase immobilization of soluble FcεRIα on ELISA plates leads to asignificant reduction of its capacity to bind IgE. This effect is mostlikely due to changes in the tertiary structure of rsFcεRIα induced bythe hydrophobic interactions of the protein with the solid phase matrix.Therefore, conventional ELISA conditions are inappropriate for thedetection of serum IgG anti-FcεRIα autoantibodies. To circumvent thisproblem, an ELISA system is established that allows the binding ofautoantibodies to native, non-denatured rsFcεRIα in a liquid phase. Sucha strategy mimics the natural binding conditions of serum IgGanti-FcεRIα autoantibodies with their target in vivo. Hence, a definedmolar concentration of biotinylated rsFcεRIα is allowed to react withdiluted serum specimens, an aliquot of this reaction mixture is thentransferred to a streptavidin-bound ELISA plate and the binding, of IgGanti-FcεRIα/rsFcεRIα complexes is monitored using peroxidase-coupledanti-human IgG antibodies.

3. Mouse mAbs Directed Against the Fc Part of Human IgE are Required toEliminate the Interference of Serum IgE with rsFcεRIα

Serum IgE binds rsFcεRIα and therefore can (I) competitively inhibitautoantibody-binding to rsFcεRIα and/or (II) allow the binding ofnaturally occurring IgG anti-IgE antibodies to rsFcεRIα. To avoid thisIgE-mediated interference with the ELISA system, sera are preincubatedwith mouse mAbs directed against the Fe part of human IgE prior to theirexposure to biotinylated rsFcεRIα. Using the anti-IgE mAb BSW17 acomplete blockage of serum IgE binding to rsFcεRIα is observed.Importantly, preincubation of sera with this mAb does not at allinfluence the binding of serum IgG anti-FcεRIα to rsFcεRIα. Therefore,mAb BSW17 is routinely added to the serum specimens prior to theanalysis. Scheme of the ELISA method is given in FIG. 3.

4. Methodology

Serum dilutions (1:100; 1:400) are incubated with 2 nM mAb BSW17 for atleast 3 hours at room temperature. Thereafter, half of the sample volumeis removed and incubated with non-biotinylated rsFcεRIα overnight at 4°C. All samples are then reacted with 5 nM biotinylated rsFcεRIα for 3hours at 37° C. 100 μl aliquots of these reaction mixtures aretransferred to ELISA plates covalently coupled to streptavidin andincubated for 1 hour at room temperature. After several rounds ofwashings, plates are reacted with peroxidase-coupled anti-human IgGF(ab')₂ (1:10000) or anti-human IgE. The amount of plate-bound enzymewas evaluated using TMB.

5. Results

mAb BSW17 blocks the interference of serum IgE with soluble recombinantFcεRIα. In order to apply serum rather than purified serum IgG forroutine diagnostics of IgG anti-FcεRIα autoreactivity, the possibleinterference of serum IgE with the ELISA system had to be eliminated.Therefore, the anti-human IgE mAb BSW17 was tested for its ability toblock IgE binding to rsFcεRIα. Serum from atopic dermatitis (AD;containing >10⁴ U IgE) and from chronic urticaria (CU) patients wereanalyzed for their IgE and IgG reactivity with rsFcεRIα in the presenceor absence of mAb BSW17. As depicted in FIG. 4, preincubation of AD serawith BSW17 abolished the rsFcεRIα-bound IgE reactivity. In contrast, thersFcεRIα-bound IgG reactivity of CU sera was not affected by mAb BSW17.Virtually identical results were obtained when BSW17-exposed sera wereused in immunoblotting experiments or when 2 other anti-IgE mAbs wereused. Taken together, these results show that preincubation of sera withBSW17 abrogates the possible interference of serum IgE and thus shouldallow the detection of IgG anti-FcεRIα autoantibodies in unfractionatedserum samples.

Specificity of the rsFcεRIα-Based ELISA Detection System

Next it was investigated whether (i) serum biotin, (ii) the serum IgGconcentration, or (iii) insect cell-expressed recombinant proteins otherthan rsFcεRIα can interfere with the ELISA detection of IgG anti-FcεRIαautoantibodies. (I) Preincubation of streptavidin-coated platesovernight with various concentrations of healthy donors' serum neitherquantitatively nor qualitatively affects the detectability ofautoantibodies (FIG. 5A). However, incubation with pure serum results instronger signals, explainable by enhanced background. Nevertheless, thepossibility that soluble serum-derived biotin may inhibit IgGanti-FcεRIα/rsFcεRIα complex-binding to streptavidin-coated ELISA platescan be ruled out. (ii) When increasing concentrations of irrelevant(i.e., IgG anti-FcεRIα-negative sera) are mixed withautoantibody-containing sera no influence on the detectability of IgGanti-FcεRIα autoantibodies is observed (FIG. 5B). This indicates thatthis system allows the reliable detection of autoantibodies irrespectiveof the IgG concentrations of the serum specimen tested. (iii) In orderto test the antigen specificity of this ELISA test system. sera withvarious concentrations of rsFcεRIα or e.g. recombinant L1 protein ofrhesus monkey papilloma virus (rRhPV) are preincubated prior toanalysis. The experiments reveal that selectively rsFcεRIα but not rRhPVcan inhibit the interaction of serum IgG anti-FcεRIα autoantibodies withbiotinylated rsFcεRIα (FIG. 5C). These findings give an additional prooffor the specificity of this test system and exclude that the observedreactivity is due to serum IgG reactivity to insect sugar components. Infurther studies, non-biotinylated rsFcεRIα protein in concentrationsequimolar to those of biotinylated rsFcεRIα was applied.

Results obtained indicate that the ability to block ELISA reactivity byhigh concentrations of rsFcεRIα can be used to discriminate IgGanti-FcεRIα autoantibody-containing and non-containing serum samples.Thus, besides the OD value reflecting the IgG-binding to the ELISAplates, the ability to block this reactivity by non-labeled RsFcεRIα isa valuable indicator for the presence of IgG anti-FcεRIα autoantibodies(FIG. 6).

Correlation of Results Obtained in Western Blot (WB) and ELISA Analysis

The comparative analysis of results obtained in WB studies and ELISAexperiments reveals good correlation between these two methods. FIG. 7shows that WB-reactive sera also exhibit ELISA reactivity in 7 out of 8cases. None of the WB-negative samples shows reactivity in the ELISAdetection system. Sera that cannot be classified in WB due to highbackground staining, show either weak or no reactivity in the ELISAindicating that these specimens may contain low titer autoantibodylevels not discernible in WB analysis.

Patient Study

In an extended patient study the presence of IgG anti-FcεRIαautoreactivity in 85 patients suffering from chronic urticaria and 20atopic dermatitis patients versus 31 healthy controls was evaluated.Results are shown in FIG. 8. IgG anti-FcεRIα autoreactivity is foundpredominantly if not exclusively in the group of CU patients.

What is claimed is:
 1. An assay for high capacity screening forsubstances that interfere with the binding of human IgE to its highaffinity receptor FcεRIα comprising admixing a solution containing asubstance to be tested with dissolved, biotinylated Fcε RIα receptor toform a mixture; admixing the mixture with IgE to form a binding reactionmixture; transferring the binding reaction mixture to astreptavidin-coated multiple well plate; and detecting whether thesubstance to be tested interferes with binding of IgE to biotinylatedFcε RIα receptor in the binding reaction mixture by means of anenzyme-labeled antibody raised against human IgE.
 2. A method for thedifferential analysis between autoimmune disorders and allergiestriggered by exogenous factors in a patient, a serum sample having beenobtained from the patient, comprising determining if IgG that bind tothe Fcε RIα receptor are present in the serum sample from the patientby: admixing the serum sample with dissolved, biotinylated Fcε RIαreceptor to form a binding reaction mixture; transferring the bindingreaction mixture to a streptavidin-coated multiple well plate; anddetecting the presence of IgG that bind to the biotinylated Fcε RIαreceptor in the binding reaction mixture by means of an enzyme-labeledantibody raised against human IgG, wherein the presence in the patientserum sample of IgG that bind to the biotinylated Fcε RIα receptorindicates that the patient is suffering from an autoimmune disorderrather than an allergy triggered by exogenous factors.
 3. The method ofclaim 2, which further comprises, prior to determining if IgG that bindto the FcεRIα receptor are present in the serum sample from the patient,the step of preincubating the serum sample from the patient with ananti-IgE antibody.
 4. The method of claim 3, wherein said anti-IgEantibody is the antibody BSW17 produced by the hybridoma cell linedeposited with the European Collection of Animal Cell Cultures under theprovisions of the Budapest Treaty on the deposit of microorganisms underdeposit number 96121916.